This application addresses Broad Challenge Area (06) Enabling Technologies, Topic 06-GM-105 Small RNAs. RNA interference (RNAi) is a post-transcriptional gene silencing mechanism mediated by small interfering RNA (siRNA) or microRNA (miRNA). The significance of RNAi is underscored by its wide existence throughout metazoans and fundamental roles in biology and disease. The catalytic engine of RNAi is the RNA-induced silencing complex (RISC), which directs sequence-specific cleavage of target mRNA. A minimal RISC can form between recombinant Ago2, the ribonuclease of RISC, and single stranded siRNA, but not with double-stranded (ds) siRNA. In Drosophila, Dicer2 and R2D2 coordinately recruit nascent duplex siRNA to Ago2 to promote RISC assembly. Reconstitution of holo-RISC presents a major challenge in the RNAi field. The goal of this proposal is to understand Drosophila RISC using a biochemical fractionation and reconstitution approach. In Aim 1, we reconstituted long dsRNA-and duplex siRNA-initiated RISC activities using purified recombinant Dicer2/R2D2/Ago2 proteins. Furthermore, this core RISC reconstitution system was used to purify a novel RNAi activator that consists of Translin and Trax proteins. We will understand the mechanism by which Translin/Trax complex enhances the assembly and activity of Drosophila RISC. In Aim 2, we discovered that the RISC loading complex (RLC), whose formation precedes and is essential for RISC assembly, contained unknown factor(s) besides Dicer2/R2D2 and siRNA. We will purify this unknown factor(s), which we named as RLC-X, by chromatographic fractionation. We will perform genetic and biochemical reconstitution studies to understand exactly how the formation of RLC facilitates the assembly of RISC. This series of in-depth studies will significant advance our understanding of the composition, assembly, and regulation of holo- RISC, the catalytic engine of RNAi. The knowledge will greatly facilitate development of novel therapeutics for human diseases. The origins of human diseases, such as cancer, can be generally attributed to loss-of-function of important genes (tumor suppressor genes) and/or gain-of-function of pathological genes (oncogenes). We are interested in understanding how animal cells use small RNAs to silence target gene expression. Our studies will not only advance understanding of the critical roles of small RNAs in biology and disease, but also facilitate development of novel therapeutics for human disease by using small RNAs to specifically shut down the expression of pathological genes.